Recombinant antibodies have the capability to specifically identify protein targets and are now a successful class of therapeutics. The success of antibodies in the clinical setting both validate their ability to bind to cell surface targets i vivo and open the possibility of using these constructs as non-invasive imaging agents. Engineered antibodies with modified pharmacokinetics have been utilized and shown to accumulate rapidly in tumors against target antigens while clearing rapidly from the blood, allowing imaging on the same or next day. Single chain variable fragments (scFvs) are created by joining antibody variable light and variable heavy chains via a peptide linker and can be identified using phage display technologies. However, the use of these engineered antibodies may result in a reduced immunoreactivity after the random conjugation of chelating agents for radiolabeling with radioactive metals. In general, conjugation of bifunctional chelating agents (BCAs) to an antibody or antibody construct occurs randomly through existing amino acid side chains in the protein. In particular, coupling occurs through lysine side chains (-amino group) or cysteines (sulfhydryl). This leads to heterogeneous coupling which can lead to inactivation of the construct and a nonhomogeneous mixture of products that are difficult to characterize biochemically. Thus, we propose to engineer unnatural amino acids (UAAs) into scFv constructs such that BCAs can be chemically conjugated in a site-specific manner. Previous studies have shown that UAAs can be incorporated into a Fab fragment of trastuzumab by supplementing the media of cells containing engineered tRNA synthetases which then incorporates the UAA at a site-specific amber codon. In these studies we will use an anti-ubiquitin scFv as a model system and the Methanococcus jannaschii tyrosyl-tRNA synthetase that is specific for insertion of p-azido-phenylalanine. This will be used to determine sites within the scFv that are amenable to click conjugation to p-azido-phenylalanine without disrupting immunoreactivity. We will then modify the M. jannaschii tyrosyl-tRNA synthetase such that it recognizes a novel UAA (-azido-lysine) that is more versatile for future click reactions. This will be evaluated for incorporation into th anti-ubiquitin scFv and the chemistry compared to the p-azido- phenylalanine constructs. The specific aims of the proposal are 1.) Construct an anti-ubiquitin scFv that contains p-azido-phenylalanine at specific sites and evaluate after conjugation to a BCA and 2.) Modify 26tyrosyl-tRNA synthetase such that it can incorporate a novel UAA into specific sites in scFvs. Overall, these studies will provide a methodology for incorporation of these two UAAs into any scFv such that a BCA or other agent can be conjugated to the scFv without loss of immunoreactivity.